Full Genotyping Research Articles

The frequency of HLA-DQ7 in patients at risk of coeliac disease: A haplotype to be reckoned with for screening?

HLA-DQ2 and -DQ8 carriers are genetically predisposed to develop coeliac disease (CD). Testing negative for HLA-DQ2/DQ8 has a high negative predictive value. Full HLA-DQ genotyping as well as tests only typing for HLA-DQ2/DQ8 are therefore used for screening at-risk populations. However, HLA-DQ7 positive and HLA-DQ2/DQ8 negative CD patients have been described in various populations. In this study we examine the relevance for HLA-DQ7 typing in a large at-risk CD population.The HLA-DQ status of all paediatric and adult patients at-risk of CD that were typed in a tertiary medical centre laboratory between 2012 and 2016 (n = 3983) was obtained. HLA-DQ7 (HLA-DQB1*03:01) positive and HLA-DQ2/DQ8 negative patients were selected. We gathered information on serology, histology and dietary status, and CD diagnosis.In total, 489/3983 patients were HLA-DQ7-positive and HLA-DQ2/DQ8 negative, and after exclusion (missing data on diet or serology/histology), 325 were included. Only one adult patient was diagnosed with CD, based on a duodenal biopsy and a clinical response to a gluten-free diet. Homozygosity was observed in 14.8 %.Based on the current cohort additional typing of HLA-DQ7 does not seem relevant for screening at-risk populations for CD in the Netherlands. It should be considered in patients with a high suspicion of CD.

Development, Validation, and Implementation of an Augmented Multiwell, Multitarget Quantitative PCR for the Analysis of Human Papillomavirus Genotyping through Software Automation, Data Science, and Artificial Intelligence

The value of HPV testing for cervical cancer screening is well established, where its use as primary screening option or as reflex test after atypical cytology results has recently gained wide acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management.In this study a new analysis method for the RIATOL quantitative PCR assay was developed, validated and implemented in the laboratory of clinical molecular pathology at A.M.L. (Sonic Healthcare, Belgium), under national accreditation and following the international ISO guidelines. It presents the successful validation of a high throughput, multi-target HPV analysis method, with enhanced accuracy on both qualitative and quantitative end-results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence (DS/AI). Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources.Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL qPCR assay proved to be a remarkable advancement in high throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Comparative peculiarities of genomic diversity in Gallus gallus domesticus chickens with decorative plumage: the muffs and beard phenotype.

Throughout history, humans have been attempting to develop the ornamental features of domestic animals in addition to their productive qualities. Many chicken breeds have developed tufts of elongated feathers that jut out from the sides and bottom of the beak, leading to the phenotype known as muffs and beard. It is an incomplete autosomal dominant phenotype determined by the Mb locus localised on chromosome GGA27. This project aimed to analyse the genetic diversity of chicken breeds using full genomic genotyping with the Chicken 60K BeadChip. A total of 53,313 Single Nucleotide Polymorphisms were analysed. DNA was obtained from breeds with the muffs and beard as a marker phenotype: Faverolles (n = 20), Ukrainian Muffed (n = 18), Orloff (n = 20), Novopavlov White (n = 20), and Novopavlov Coloured (n = 15). The Russian White (n = 20) was selected as an alternative breed without the muffs and beard phenotype. The chickens are owned by the Centre of Collective Use "Genetic Collection of Rare and Endangered Breeds of Chickens" (St. Petersburg region, Pushkin), and are also included in the Core Shared Research Facility (CSRF) and/or Large-Scale Research Facility (LSRF). Multidimensional scaling revealed that the Novopavlov White and the Novopavlov Coloured populations formed a separate group. The Ukrainian Muffed and the Orloff have also been combined into a separate group. Based on cluster analysis, with the cross-validation error and the most probable number of clusters K = 4 taken into account, the Orloff was singled out as a separate group. The Ukrainian Muffed exhibited a notable similarity with the Orloff under the same conditions. At K = 5, the populations of the Novopavlov White and the Novopavlov Coloured diverged. Only at K = 6, a distinct and separate cluster was formed by the Ukrainian Muffed. The Russian White had the greatest number of short (1-2 Mb) homozygous regions. If the HOXB8 gene is located between 3.402 and 3.404 Mb on chromosome GGA27, homozygous regions are rarely found in the chickens with the muffs and beard phenotype. Scanning the chicken genome with the Chicken 60K BeadChip provided enough information about the genetic diversity of the chicken breeds for the peculiarities of the development of the ornamental muffs and beard phenotypes in them to be understood. For example, Phoenix bantams, whose tail feathers grow throughout their lives, require greater consideration of husbandry conditions.

PMSeeker: A Scheme Based on the Greedy Algorithm and the Exhaustive Algorithm to Screen Low-Redundancy Marker Sets for Large-Scale Parentage Assignment with Full Parental Genotyping.

Parentage assignment is a genetic test that utilizes genetic characteristics, such as molecular markers, to identify the parental relationships within populations, which, in commercial fish farming, are almost always large and where full information on potential parents is known. To accurately find the true parents, the genotypes of all loci in the parentage marker set (PMS) are required for each individual being tested. With the same accuracy, a PMS containing a smaller number of markers will undoubtedly save experimental costs. Thus, this study established a scheme to screen low-redundancy PMSs using the exhaustive algorithm and greedy algorithm. When screening PMSs, the greedy algorithm selects markers based on the parental dispersity index (PDI), a uniquely defined metric that outperforms the probability of exclusion (PE). With the conjunctive use of the two algorithms, non-redundant PMSs were found for more than 99.7% of solvable cases in three groups of random sample experiments in this study. Then, a low-redundancy PMS can be composed using two or more of these non-redundant PMSs. This scheme effectively reduces the number of markers in PMSs, thus conserving human and experimental resources and laying the groundwork for the widespread implementation of parentage assignment technology in economic species breeding.

Search for genetic markers for selection and breeding aimed at increasing birth weight of piglets

With the intensive increase in the fertility traits of sows, the problem of decreasing the weight of piglets at birth has arisen. In this connection, the search for genetic variants associated with the birth weight of piglets is of particular relevance and scientific significance. The aim of the work was to identify genetic variants associated with piglet weight at birth and test them to select optimal genetic markers for selection and breeding work to improve reproductive performance of pigs on the basis of full genomic genotyping data using the Fst method. The studies were conducted in 2020-2022 on Large White pigs (n=239) bred at CJSC Plemzavod-Yubileiny in the Tyumen Region. Genotyping was performed using GeneSeek® GGP Porcine HD Genomic Profiler v1 (Illumina Inc., USA). Genomic data were filtered according to the following parameters --geno 0.1, -mind 0.1, -maf 0.05, -hwe 1e-7, --indep-pairwise 50 5 0.8. To identify genomic regions associated with piglet birth weight, there was used Fst statistics comparing genetic variants in pigs between two groups with high and low indices. Those in which the Fst values exceeded the quantile level of 0.999 were considered significant variants. Student's test was used to evaluate the significance of the effects of variant genotypes on the birth weight and number of piglets at birth. The results showed that there was a moderate negative relationship (-0.351) between piglet birth weight and number of piglets at birth. 17 SNPs associated with birth weight of piglets were identified, 9 of which were located in the KIF13A, STK24, FDFT1, ADGRD1, STX2, TMEM132D, ENSSSCG00000054866, ENSSSCG00000058459 genes, as well as SNPs rs81450496, rs80887103 in intergenic regions have been identified as promising genetic markers for increase in birth weight of piglets. The results obtained can be used to create domestic breeding technologies that improve the efficiency of pig breeding.

TOP-103 - HDV full genome sequencing and sensitive HBV genotyping from a large cohort of HBV/HDV co-infected patients

TOP-103 - HDV full genome sequencing and sensitive HBV genotyping from a large cohort of HBV/HDV co-infected patients

Contemporary biomarker testing rates in both early and advanced NSCLC: Results from the MYLUNG pragmatic study.

9109 Background: The MYLUNG (Molecularly Informed Lung Cancer Treatment in a Community Cancer Network) consortium pragmatic study aims to identify and overcome barriers to timely and appropriate biomarker testing in patients (pts) with NSCLC. We previously reported retrospective real-world testing rates for 5 biomarkers from 2018-2020 in metastatic pts. We now report results from the prospective observational phase, including additional biomarkers in pts with both early and advanced NSCLC. Methods: This was a prospective, non-interventional cohort study of newly diagnosed pts with early stage (ES), locally advanced (LA), or metastatic NSCLC (mNSCLC) between 12/2020 and 9/2022 and enrolled from 12 community practices (69 sites) across The US Oncology Network. The proportion of pts with test results for 9 biomarkers at time of protocol initiation were examined: ALK, BRAF, EGFR, ROS1, PD-L1, KRAS, MET, NTRK, and RET. Timing of tests, use of single vs multigene NGS testing, clinical and socioeconomic factors, and reasons for not testing were collected in real time. Results: Of 1002 pts screened, 987 were enrolled and analyzed. There were 405 stage IB-IIIC pts [n = 284 stage IB-IIIA (ES cohort); n = 121 stage IIIB-IIIC (LA cohort)] and 582 stage IV pts (mNSCLC cohort). Descriptions for ES and LA vs mNSCLC pts respectively include median age 68 vs 69 yrs, 49% vs 52% female, 83% vs 77% white, 58% vs 79% adenocarcinoma histology, 71% vs 68% ECOG 0-1, and 33% vs 30% high school education level. Testing rates for ES vs mNSCLC cohorts are shown in the table below: prior to treatment initiation, 64% of ES and 83% of mNSCLC pts had at least one biomarker result; and 37% of mNSCLC pts had results for all 9 biomarkers. NGS testing occurred in 37% and 57% of the ES and mNSCLC cohorts respectively. Reasons for not testing included: barriers to ordering (42%, 25%), insufficient tissue (18%, 18%), clinical deterioration/crisis (5%, 12%), and other reasons (37%, 49%), respectively. Conclusions: As more genomic alterations are identified in NSCLC, we observed that many pts do not get full genotyping prior to treatment, and biomarker testing remains of utmost importance. Further analyses by distinct stages (e.g. LA), histology, social determinants, utilization of targeted therapies, and other reasons for not testing will be performed. These data will be used to evaluate future prospective interventions for MYLUNG, to help improve comprehensive biomarker testing for NSCLC. [Table: see text]

Real-World Biomarker Testing Patterns in Patients With Metastatic Non-Squamous Non-Small Cell Lung Cancer (NSCLC) in a US Community-Based Oncology Practice Setting

Introduction/BackgroundThis study was designed to describe real-world changes in biomarker testing among patients with non-squamous, metastatic non-small cell lung cancer (mNSCLC) in a community oncology setting from 2015 to 2020. Patients and MethodsThis retrospective study randomly selected 500 adult patients diagnosed with nonsquamous mNSCLC to undergo chart review and data extraction. Data were extracted and validated by 2 independent abstractors. Biomarker testing rates were described before and after national guideline updates and FDA approval of targeted agents. ResultsAt least 1 biomarker test was received by 89.4% of patients with mNSCLC. Of all patients, 46.6%, 34.6%, and 8.2% received both single-gene and next generation sequencing (NGS)-based testing, single-gene testing only, and NGS-based testing only, respectively. However, there were changes in testing rates at the time of drug approvals for targeted agents. Biomarker testing increased for ALK (45.0% before to 78.3% after ALK-targeted drug approval), BRAF (from 20.0% to 67.8%), EGFR (from 20.0% to 78.2%), NTRK (from 34.6% to 55.7%), and ROS1 (increased from 29.6% before approval to 74.2% after). Biomarker testing increased after changes were made to national guidelines for BRAF (from 18.8% before to 68.1% after inclusion in guidelines), NTRK (from 37.2% to 56.5%), and ROS1 (increased from 40.8% to 74.5% after guideline updates). Targeted therapy was received by 62.4% of patients with a positive biomarker. ConclusionIncreases in biomarker testing rates were observed relative to targeted agent approvals and national guideline updates. However, many patients with non-squamous mNSCLC did not receive full genotyping in accordance with national guidelines and represent an opportunity to identify reasons and solutions for barriers to care.

Does full HPV genotyping perform similarly well in clinician-collected cervical samples and self-collected vaginal samples when using the CLART HPV4S assay?

BackgroundStudies comparing self-collected vaginal samples with clinician-collected cervical samples with respect to high-risk human papillomavirus (HPV) detection and genotype agreement based on clinically validated full HPV genotype assays (e.g. the CLART HPV4S) are limited. This study compared the two types of samples using the CLART assay with respect to HPV detection and genotype agreement in a referral population.MethodsA total of 212 women aged 30–59 years and diagnosed with atypical squamous cells of undetermined significance (ASC-US) within the Danish cervical cancer screening programme had a cervical sample taken at their general practitioner. Afterwards, the women took a vaginal sample with the Evalyn Brush device at home. The paired samples were HPV-tested with the full genotyping CLART HPV4S assay. Histological results, i.e. cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) were available for 14 women with HPV-positive clinician-collected samples.ResultsThe study found the same HPV prevalence in self-collected vaginal samples compared to clinician-collected cervical samples (19.3%, 95% CI 14.3–25.3% vs 18.4%, 95% CI 13.4–24.3%). The CLART HPV4S assay detected approximately the same number of CIN2+ cases in the self-collected vaginal samples compared to the clinician-collected cervical samples (13 vs 11 cases). Exactly the same genotypes were detected in 75% (21/28) of the HPV-positive paired samples, while at least one identical genotype was found in the remaining 25% (7/28) of the paired samples.ConclusionsThe CLART HPV4S assay performed similarly well in self-collected vaginal samples as in clinician-collected cervical samples with respect to both HPV detection and genotype agreement when using the Evalyn Brush and the CLART HPV4S assay in a referral population. Although further evaluation is needed, the findings suggest that full HPV genotyping based on the CLART assay can be useful when establishing HPV genotype-specific referral strategies for women tested HPV-positive by self-sampling.

Genetic predisposition to gestational diabetes mellitus in the Kazakh population

Background and aimsThe purpose of the study was to conduct a comparative analysis of population frequencies of alleles and genotypes of polymorphic variants of genes for impaired insulin synthesis and associated with insulin signal transduction. MethodsThis investigation uses a genomic database of 1800 conditionally healthy individuals of Kazakh ethnicity, who underwent full genome genotyping using OmniChip 2.5–8 Illumina chips of ∼2.5 million Single Nucleotide Polymorphism at deCODE Iceland Genomic Centre. ResultsThe highest frequency of carriage of minor A allele – 17.6% rs4607517 polymorphism of Glucokinase gene, unfavorable genotypes A/G – 29.5% and A/A – 3.0% in comparison with European and Asian populations, indicates a contribution of hereditary family forms of Maturity-onset diabetes of the young type 2 to gestational diabetes mellitus in Kazakh population. ConclusionsThe study of the associations of genetic markers of gestational diabetes mellitus will allow timely identification of high-risk groups before and at an early stage of pregnancy, carrying out the necessary effective preventive measures and, in the case of gestational diabetes mellitus development, optimizing the correction of carbohydrate metabolism disorders and predicting outcomes for the mother and the fetus.

Full genotyping and FAM19A4/miR124-2 methylation analysis in high-risk human papillomavirus-positive samples from women over 30 years participating in cervical cancer screening in Örebro, Sweden.

Currently, cervical cancer prevention is undergoing comprehensive development regarding human papillomavirus (HPV) vaccination and cervical cancer screening. In Sweden and many other countries, high coverage vaccinated cohorts are entering screening within the next few years. This entails demands for baseline HPV genotype data across the screening age range for surveillance and a basis for screening program adjustment. In 2016, Örebro County, Sweden, changed to primary HPV screening using HPV mRNA testing followed by cytology triage. An alternative triage method to cytology could allow for a fully molecular screening algorithm and be implemented in a screening program where self-sampling is included. Hypermethylation analysis of the human genes FAM19A4/miR124-2 has been suggested as a promising triage method. HPV mRNA-positive screening samples (n = 529) were included and subjected to genotyping targeting a broad range of both low-risk and high-risk genotypes in addition to hypermethylation analysis of the two human genes FAM19A4/miR124-2. Data were connected to cytological and histological status and age. The most commonly detected genotypes were HPV31, 16, and 52. In addition, HPV18 was one of the most common genotypes in high-grade squamous intraepithelial lesions (HSILs) samples. In relation to available vaccines, 26% of the women with histological HSIL or cancer (≥HSIL) tested positive for only hrHPV included in the quadrivalent vaccine and 77% of the genotypes in the nonavalent vaccine. According to these figures, a relatively large proportion of the HSILs will probably remain, even after age cohorts vaccinated with the quadrivalent vaccine enter the screening program. Hypermethylation positivity was associated with increasing age, but no HPV-related independently predictive factors were found. Accordingly, age needs to be considered in development of future screening algorithms including triage with hypermethylation methodology.

Evaluation of the MHSeqTyper47 kit for forensically challenging DNA samples

Microhaplotypes have been highly regarded for forensic mixture DNA deconvolution because they do not experience interference from stutters in the same way as short tandem repeat markers, and they tend to be more polymorphic than single nucleotide polymorphism markers. However, forensic microhaplotype kits have not been reported. The MHSeqTyper47 kit genotypes 47 microhaplotype loci. In this study, MiSeq FGx sequencing metrics for MHSeqTyper47 were presented, and the genotyping accuracy of this kit was examined. The sensitivity of MHSeqTyper47 reached 62.5 pg, and full genotyping results were obtained from degraded DNA samples with degradation indexes ≤ 3.00. Full genotypes were obtained in the presence of 100 ng/μL tannin, 50 μM heme, 25 ng/μL humic acid, and 1.25 μg/μL indigo dye. In DNA mixture studies, a minimum of 31 loci of the minor contributor were correctly genotyped at 1:99 or 99:1 mixing ratios, with the cumulative random matching probability of these loci reaching 4.54 × 10−25. Mixing ratios could be reliably predicted from two-donor DNA mixtures based on the loci with four called alleles. Taken together, these data showed that the MHSeqTyper47 kit was effective for forensically challenging DNA analysis.

Comparison of Seegene Anyplex II HPV28 assay with BD Onclarity HPV assay for human papillomavirus genotyping.

Presently, human papillomavirus (HPV)-based cervical cancer screening is commonly used and is replacing conventional cytology screening tests. The HPV genotyping assay is useful for triage in cervical cancer screening and the evaluation of HPV vaccination effects. In this study, we evaluated the clinical performance of two HPV genotyping assays, BD Onclarity HPV (Onclarity) and Seegene Anyplex II HPV28 (Anyplex) in the detection of relevant cervical lesions and for HPV genotyping concordance. Anyplex and Onclarity assays were performed on 920 consecutive liquid-based specimens. Anyplex, sensitivity, specificity, and genotyping concordance with Onclarity were optimal when restricted to ≥2+ (medium) viral loads. HPV genotyping agreement between the two assays ranged between 0.75 and 0.9 (excellent), except for HPV 33/58, which was 0.73 (good). With Onclarity as a reference, the relative sensitivity of Anyplex for the detection of ≥CIN 2 was 1.05 (95% CI: 0.99–1.1) and the relative specificity for detection of negative for intraepithelial lesion and malignancy (NILM) was 0.89 (95% CI: 0.85–0.93). For most ≥CIN 2 lesions, high-risk HPV was detected by Onclarity (66/72) and Anyplex (69/72) assays. For high-risk HPV negative ≥CIN 2 lesions, possible high-risk HPV genotypes were detected by Anyplex. In conclusion, the genotyping agreement between the tests was good to excellent. Full genotyping with Anyplex might confer additional benefits to patients with ≥CIN 2, although the difference is small. We also suggest an optimal cutoff value when reporting HPV infections using the Anyplex assay (≥2+; medium viral loads).

Typing of semen-containing mixtures using ARMS-based semen-specific CpG-InDel/STR markers.

Mixed traces are common biological materials found at crime scenes, and their identification remains a significant challenge in the field of forensic genetics. In recent years, DNA methylation has been considered as a promising approach for body fluid identification, and length polymorphic loci are still the preferred markers for personal identification. In this study, we used tissue-specific CpG sites with linked insertion or deletion (InDel) or short tandem repeat (STR) markers (CpG-InDel/STR) for both body fluid and individual identification. The tissue-specific CpG loci, which were all selected from the previous reports, were analyzed using a combination of bisulfite conversion and amplification refractory mutation system-multiprimer-PCR technology. InDels or STRs, which were selected within 400bp upstream or downstream of the semen-specific CpG loci, were analyzed using a capillary electrophoresis platform. Eventually, we successfully constructed a panel containing 17 semen-specific CpG-InDel/STR compound markers compassing 21 InDels/STRs, 3 body-fluid positive controls (vaginal secretion-, saliva-, and blood-specific CpG), and 1 gender identification locus. Using this panel, full genotyping of individuals could be obtained successfully with 50ng DNA input. Semen stains stored at room temperature for 7months and degraded samples that were heat treated for up to 6h were still identified efficiently. For semen containing mixed stains, it is also useful when the semen content is as low as 3.03%. Moreover, the cumulative discrimination power of this panel is 0.9999998. In conclusion, it is a robust panel enabling the validation of both the tissue source and individual identification of semen containing mixed stains and can be employed as an alternative solution for forensic case investigation.

Clinical validation of full HR-HPV genotyping HPV Selfy assay according to the international guidelines for HPV test requirements for cervical cancer screening on clinician-collected and self-collected samples

BackgroundAccording to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed – Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples.MethodsFor each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO—National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture®2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test.ResultsHPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population.ConclusionsHPV Selfy fulfills all the requirements of the international Meijer’s guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling.Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/2018

Population Genetic Structure and Hybridization of Schistosoma haematobium in Nigeria.

Background: Schistosomiasis is a major poverty-related disease caused by dioecious parasitic flatworms of the genus Schistosoma with a health impact on both humans and animals. Hybrids of human urogenital schistosome and bovine intestinal schistosome have been reported in humans in several of Nigeria’s neighboring West African countries. No empirical studies have been carried out on the genomic diversity of Schistosoma haematobium in Nigeria. Here, we present novel data on the presence and prevalence of hybrids and the population genetic structure of S. haematobium. Methods: 165 Schistosoma-positive urine samples were obtained from 12 sampling sites in Nigeria. Schistosoma haematobium eggs from each sample were hatched and each individual miracidium was picked and preserved in Whatman® FTA cards for genomic analysis. Approximately 1364 parasites were molecularly characterized by rapid diagnostic multiplex polymerase chain reaction (RD-PCR) for mitochondrial DNA gene (Cox1 mtDNA) and a subset of 1136 miracidia were genotyped using a panel of 18 microsatellite markers. Results: No significant difference was observed in the population genetic diversity (p > 0.05), though a significant difference was observed in the allelic richness of the sites except sites 7, 8, and 9 (p < 0.05). Moreover, we observed two clusters of populations: west (populations 1–4) and east (populations 7–12). Of the 1364 miracidia genotyped, 1212 (89%) showed an S. bovis Cox1 profile and 152 (11%) showed an S. haematobium cox1 profile. All parasites showed an S. bovis Cox1 profile except for some at sites 3 and 4. Schistosoma miracidia full genotyping showed 59.3% of the S. bovis ITS2 allele. Conclusions: This study provides novel insight into hybridization and population genetic structure of S. haematobium in Nigeria. Our findings suggest that S. haematobium x S. bovis hybrids are common in Nigeria. More genomic studies on both human- and animal-infecting parasites are needed to ascertain the role of animals in schistosome transmission.

Development and Validation of a Novel and Fast Detection Method for Cannabis sativa: A 19-Plex Short Tandem Repeat Typing System.

In recent years, influenced by the legalization of Cannabis sativa in some countries and regions, the number of people who smoke or abuse C. sativa has continuously grown, cases of transnational C. sativa trafficking have also been increasing. Therefore, fast and accurate identification and source tracking of C. sativa have become urgent social needs. In this study, we developed a new 19-plex short tandem repeats (STRs) typing system for C. sativa, which includes 15 autosomal STRs (D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, CS1, ANUCS 305, 3735, and ANUCS 302 and 9043), two X-chromosome STRs (ANUCS 501 and 1528), one sex-determining marker (DM016, on Y-chromosome), and a quality control marker (DM029, on autosome). The whole polymerase chain reaction (PCR) process could finish within 1 h, making the system suitable for fast detection. The PCR products were detected and separated with an Applied Biosystems 3500XL Genetic Analyser. Developmental validation studies indicated that the 19-plex typing system was accurate, reliable and sensitive, which could also deconvolute mixed C. sativa samples. Specifically, the sensitivity study showed that a full genotyping profile was obtainable with as low as 125 pg of C. sativa DNA. The species specificity study demonstrated that this multiplex has no cross-reactivity with common non C. sativa DNA. In the population study, a total of 162 alleles at 15 autosomal STRs and 14 alleles at two X-chromosome STRs were detected among 85 samples. The efficiency parameters, including the total discrimination power (TDP) and the combined power of exclusion (CPE) of the system, were calculated to exceed 0.999 999 999 999 988 and 0.998 455 889 684 078, respectively, further proving that the system could meet the needs of individual identification. To the extent of the known studies, this is the first study that included the C. sativa sex-determining marker. In conclusion, the developed new 19-plex STR typing system can successfully achieve the purposes of species identification, gender determination, and individual identification, which could be a powerful tool in tracing trade routes of particular drug syndicates or dealers or in linking certain C. sativa to a crime scene.

Evaluation of toll-like receptor 4 polymorphism in patients with hepatitis c virus-induced hepatocellular carcinoma

Background: Liver related pathologies including Hepatocellular Carcinoma (HCC) is a universal problem. Innate immunity receptors were accused in the etiopathogenesis of HCC with many conflicts. TLR4 is one of pathogen recognition receptors involved in the pathogenesis of many diseases and malignancies. TLR4 receptor polymorphisms were investigated in HCV related morbidities along with inconclusive results Objectives: to study the role of TLR4 rs 2149356 and rs 1927914 genotypes polymorphisms in HCV related HCC development. Methodology: 200 Chronically infected HCV patients were enrolled in this study. they were divided according to lab and clinical data into 100 CHC group and 100 HCC patients who were compared to health individual. The blood samples obtained were further proceed to full lab and TLR4 genotyping by RFLP-PCR technique Results: GT genotype and T allele of TLR4 rs 2149356 at 95% CI of 0.38 (0.21-0.70) was significantly increased in control group than in HCC and CHC groups. At 0.32(0.17-0.63) TLR4 rs 1927914 C allele and CT genotype was significantly increased in Controls than diseased groups while T allele is significantly increased in HCC than control group. Conclusions: TLR4 genotypes may play a protective role against HCC development among chronic HCV patients.

Clinical and analytical performance of the CLART HPV 4S assay with SurePath screening samples from the Danish cervical cancer screening program using the VALGENT framework

The CLART HPV4S (CLART4S) is a novel full genotyping assay, based on PCR/microarray technology. We assessed the clinical accuracy of the CLART4S assays under the fourth installment of the VALGENT framework. The VALGENT cohort comprised 998 consecutive cervical samples from women participating in the Danish screening programme enriched with 297 samples with abnormal cytology (100 ASCUS, 100 LSIL, 97 HSIL). The CLART4S assay detects 16 HPV genotypes individually: 14 oncogenic and two non-oncogenic HPV types. The GP5+/6+ PCR Enzyme-Immuno-Assay (GP-EIA) and GP5+/6+ PCR with Luminex genotyping (GP-LMNX) were used as comparator tests for clinical accuracy and HPV genotype concordance, respectively. The sensitivity for ≥ CIN2 for the CLART4S assay was 96.7 % (GP-EIA: 92.6 %) with a relative sensitivity of 1.04 (1.00−1.09). The sensitivity for ≥ CIN3 was 98.8 % (GP-EIA: 94.0 %), with relative sensitivity of 1.05 (1.00−1.10). The specificity for <CIN2 was 88.6 % (GP-EIA: 89.2 %) with a relative specificity of 0.99 (0.98−1.01). The CLART 4S was found to be non-inferior to that of GP-EIA for both sensitivity (p < 0.0001) and specificity (p = 0.0452). The overall oncogenic HPV concordance between CLART4S and GP-LMNX was high, however when looking at individual genotype agreement the concordance was more diverse, with the highest agreement found in the Screening population.

Vitamin D receptor gene polymorphisms and risk of hepatocellular carcinoma in hepatitis C-related liver cirrhosis

BackgroundHCV is a major risk factor for HCC; however, the exact mechanism of hepatocarcinogenesis is still not fully understood. Host genetic factors have been reported to play a significant role. Experimental studies support the tumor inhibitory effect of vitamin D on HCC cells. Several single nucleotide polymorphisms (SNPs) have been depicted in the vitamin D receptor (VDR) gene. We aimed to assess whether any of these polymorphisms could be significantly associated with increased risk of HCC.ResultsThis study was conducted on 76 patients with HCV-related liver cirrhosis (48 patients had HCC on top of cirrhosis, and the other 28 had liver cirrhosis only). All patients underwent full medical history assessment, clinical examination, laboratory investigations, abdominal ultrasonography, and genotyping of the VDR gene. HCC patients had a significantly higher frequency of ApaI CC genotype compared with those patients without HCC. There is no statistically significant difference between the studied groups at any TaqI genotypes, but the carriage of the ApaI CC genotype had a significant association with liver disease severity in both patients groups compared with ApaI CA/AA genotypes. The carriage of the ApaI CC genotype was an independent predictor for HCC in HCV-related liver cirrhosis.ConclusionsVDR ApaI polymorphism is significantly associated with the development of HCC; thus, ApaI CC genotype could be used as an important molecular marker to predict the risk of HCC in patients with HCV-related liver cirrhosis.